Bulk and single-cell RNA-seq reveal the sexually dimorphic expression pattern of dmrtb1 in zig-zag eel (Mastacembelus armatus)

Sequential hermaphroditism, whereby the sex of one individual changes during its development, has been described in many fish species. Yet, the genetic regulation of this dynamic process of sexual differentiation is not well understood. Here, we report the characterization of intersexual differences in an aquacultural fish, the zig-zag eel (Mastacembelus armatus), based on 4 years of artificial breeding. All M. armatus individuals initially developed as females, with approximately half of them then becoming males after going through an intersex phase. A DM-domain candidate gene, dmrtb1, was identified from gonadal transcriptome data screening. The expression of dmrtb1 was restricted to testis, similar to dmrt1, which is one of the conserved sex-determining genes in vertebrates. Interestingly, dmrtb1 was expressed at the onset of sex change (early stage), and gradually increased in expression in the intersex gonads until the late stage of differentiation. Dmrtb1 was also expressed in the seminiferous lobules containing cysts with germ cells. A mixed cell population from ovotestis was identified by single-cell RNA sequencing, and included mitotic fetal germ cells, meiotic prophase fetal germ cells, and gonad endothelial cells. Spermatocyte and oocyte marker genes were both enriched in the same gonadal cell population, indicating this cell population might have the potential of bisexual differentiation. The correlation between dmrtb1 expression and sex-change development suggests M. armatus as an emerging model to study the molecular mechanism of sexual differentiation in fish. Such information will be vital for promoting the conservation of this species in the wild as well as for developing a sustainable aquacultural industry.